The present invention relates to novel molecules, which can be used to measure non-transferrin-bound iron. Specifically, the molecules of the present invention can be used to diagnose disorders associated with abnormal levels of free iron, such as hereditary hemachromatosis.
The presence of non-transferrin-bound iron (NTBI) in the circulation is a pathological phenomenon, which occurs in patients with iron-overload conditions. NTBI is absent from healthy individuals where virtually all of the serum iron is bound to the iron-carrier protein, transferrin. However, in iron-overloaded individuals, the iron binding capacity of transferrin is overwhelmed, resulting in the adsorption of the excess iron to various proteins and possibly other molecules in the serum. This iron so adsorbed is collectively referred to as NTBI.
Generally, chronic iron-overload accompanied by NTBI occurs as a result of pathological conditions associated with specific diseases. Illustrative examples of such conditions include: 1) repeated transfusions, which are required by patients with various hemolytic diseases, hemoglobinopathics (among which the most common is thalassemia) or other forms of anemia whose treatment demands blood transfusions and/or iron infusion (e.g. dialysis patients) and 2) ant inherited defect causing excess iron absorption, called Hereditary Hemachromatosis. Transient, reversible NTBI can also appear in the circulation of patients undergoing chemotherapy, heart bypass operations and other conditions where large amounts of iron, such as from hemoglobin catabolism, are suddenly released into the circulation. NTBI was also found in patients receiving dialysis who are treated for anemia with erythropoietin and intravenous iron supplements.
Accurate assessment of NTBI concentration is critical for a number of therapeutic applications. Patients suffering from Iron-overload are often prescribed a regimen of Iron chelating agents, whose efficacy hinges on accurate NTBI quantification. Low-level NTBI detection is critical for accurate diagnosis of Iron-overload [Sham, et al., Asymptomatic hemochromatosis subjects: genotypic and phenotypic profiles. Blood (2000), Vol. 96: 3707–3711]. The availability of a rapid and inexpensive NTBI-test would additionally provide screening methods for populations at high-risk for from overload. Since a high frequency of genetic mutation causing Hereditary Hemachromatosis (1 in 8 are heterozygous) exists in Northern European and American populations, and a doubling of the frequency is observed in some African and African-American populations, such screening procedures would clearly impact a large segment of the population. Further, Hereditary Hemachromatosis is often initially misdiagnosed due to a lack of definitive symptoms in the first decades of life, with evidently normal transferrin-iron saturation levels.
To date, there is no single convenient and reliable method for detection of Iron-overload and for specific quantification of NTBI. Iron-overload is most often diagnosed by the estimation of total serum iron via chemical/physicochemical methods, determination of the percent transferrin-iron saturation, or serum iron-binding capacity, by measuring high-affinity binding of radioactive iron to serum components or by determining circulating ferritin levels by immunoassay. While these assays are effective in detecting severe Iron-overload, they often fail to detect low-moderate Iron overload, and additionally have been known to produce false positives.
Currently, a few methods exist for determining NTBI in biological samples. One method [Singh, S., Hider, R. C. and Porter, J. B. (1990) Anal. Biochem. 186, 320–323] utilizes HPLC isolation and incorporation of an iron chelator, deferriprone (or L1), which forms a colored complex that is stoichiometric when in contact with sample NTBI, thereby quantifying the amount of iron in the sample.
The three main drawbacks of this method are its cost, its cumbersome nature, which makes it difficult to set up in non-specialized laboratories, and its relatively low throughput efficiency.
A second method [Evans, P. J. and Halliwell, B. (1994) Methods Enzymol., 233, 82–89] employs the use of the antibiotic bleomycin, which combines with NTBI, but not with transferrin-bound iron, to form highly reactive complexes which generate DNA cleavage products. The relative amount of DNA cleavage product is proportional to the amount of input NTBI and is quantified by a thiobarbituric acid test. The method, however, tends to overestimate NTBI and may give false positive results, limiting effective clinical application.
World patent application No. 00/36422 submitted by the applicant, incorporated fully herein by reference, describe an alternative method for determining the concentration of a free metal ion, in particular non-transferrin bound iron (NTBI), in biological samples. The method consists of incubating a sample suspected of containing NTBI with a surface coated with a polymer-conjugated form of an Iron chelator, for example, a desferrioxamine (DFO) polymer, enabling sample Iron capture by the chelator. Following Iron capture, a labeled moiety containing bound Iron that can be captured by the Iron chelator is added and the concentration of NTBI in samples tested is obtained from the change in label signal obtained with the serum sample relative to control which is regarded as nominally NTBI-free.
The above NTBI detection assay provides an effective process, however the presence of several discrete steps within the assay result in a labor-intensive process, that may not be optimal for all potential clinical settings where the precision and simplicity of the assay are critical for efficient execution.
The use of fluorescent probes in detection systems for clinical applications is highly desirable. Fluorescent probe incorporation in clinical assays typically allows for simple, high-throughput efficiency detection systems. The use of fluorescent-labeled probes in developing the aforementioned NTBI detection system has been problematic, however. Coupling of fluorescent probe to the chelator results in a probe that is sensitive, by its nature, to the environment, hence affected by color, turbidity, pH, ionic strength) of solutions utilized in the assay system, confounding assay readout results.
Further confounding issues in the accurate determination of NTBI in biological samples is poor detection of aggregated Iron, present in the samples, resulting in a lower estimation than the actual amount of NTBI in a given biological sample.
The presence of apo-Transferrin, or Transferrin not bound to Iron, in biological samples, provides yet another source of error in attempting to determine absolute levels of NTBI. Since apo-Transferrin is universally found in biological samples, except in cases of extreme iron-overload where the Transferrin is 100% iron-saturated, actual NTBI in vivo may in fact, bind to apo-Transferrin in the assay sample, resulting in a low estimation of in vivo NTBI levels.
While it is a logical consideration to utilize fluorescein-labeled Transferrin as a means of detecting NTBI within a given sample, the existing art for conjugating fluorescein to transferrin (Egan T J: Fluorescence energy transfer studies on fluoresceinated human serum transferrin. Identification of the possible fluoresceination sites. S. Aft. J. Sci. 89: 446–50) conjugated the fluorescent label to iron-loaded Transferrin. A major drawback to fluorescein-transferrin prepared by this protocol is its failure to detect iron appropriately when measures are taken to inactivate endogenous apo-Transferrin within samples, resulting in a lower level of NTBI than what is actually present in vivo. Utilization of Gallium compounds for inactivation of endogenous apo-Transferrin with this protocol result in inaccurate detection of NTBI, and hence does not provide an accurate assay system.
There is thus a widely recognized need for, and it would be highly advantageous to have, an NTBI detection assay, devoid of the above limitations. A highly sensitive assay system for the quantification of NTBI that is cost-effective, providing high-throughput efficiency, yet not compromised for accuracy and sensitivity has broad clinical application in both the diagnosis and validation of treatment regimens for Iron-overload conditions.